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Correlation spectroscopy and molecular dynamics simulations to study the structural features of proteins.

Varriale A, Marabotti A, Mei G, Staiano M, D'Auria S - PLoS ONE (2013)

Bottom Line: Our results showed that keeping temperature constant, the protein diffusion coefficient decreased from 84±4 µm(2)/s to 44±3 µm(2)/s when pH was changed from 7.0 to 4.0.An even more marked decrease of the MalE2 diffusion coefficient (31±3 µm(2)/s) was registered when pH was raised from 7.0 to 10.0.The obtained fluorescence correlation data, corroborated by circular dichroism, fluorescence emission and light-scattering experiments, are discussed together with the MD simulations results.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Molecular Sensing, IBP-CNR, Naples, Italy. a.varriale@ibp.cnr.it

ABSTRACT
In this work, we used a combination of fluorescence correlation spectroscopy (FCS) and molecular dynamics (MD) simulation methodologies to acquire structural information on pH-induced unfolding of the maltotriose-binding protein from Thermus thermophilus (MalE2). FCS has emerged as a powerful technique for characterizing the dynamics of molecules and it is, in fact, used to study molecular diffusion on timescale of microsecond and longer. Our results showed that keeping temperature constant, the protein diffusion coefficient decreased from 84±4 µm(2)/s to 44±3 µm(2)/s when pH was changed from 7.0 to 4.0. An even more marked decrease of the MalE2 diffusion coefficient (31±3 µm(2)/s) was registered when pH was raised from 7.0 to 10.0. According to the size of MalE2 (a monomeric protein with a molecular weight of 43 kDa) as well as of its globular native shape, the values of 44 µm(2)/s and 31 µm(2)/s could be ascribed to deformations of the protein structure, which enhances its propensity to form aggregates at extreme pH values. The obtained fluorescence correlation data, corroborated by circular dichroism, fluorescence emission and light-scattering experiments, are discussed together with the MD simulations results.

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Fluorescence correlation spectroscopy measurements.Autocorrelation spectra and residual distribution (inset) of MalE2 at pH 7.0 (panel A), pH 4.0 (panel B) and pH 10.0 (panel C). The best fitting of the data was obtained by the Equation 1.
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pone-0064840-g001: Fluorescence correlation spectroscopy measurements.Autocorrelation spectra and residual distribution (inset) of MalE2 at pH 7.0 (panel A), pH 4.0 (panel B) and pH 10.0 (panel C). The best fitting of the data was obtained by the Equation 1.

Mentions: The correlation function observed in the FCS measurements on MalE2-AL488 in its native state at neutral pH (panel 1A) and at pH 4.0 (panel 1B ) and 10.0 (panel 1C) is shown in Figure 1. The data were analyzed by different models and the best fitting function resulted to be Equation 1, which takes into account the possibility to have one or two species in solution. In particular, the MalE2-AL488 diffusion coefficient at pH 7 was found to be D1 = 84±4 µm2/s, a value compatible with a spherical-shaped molecule with a radius of about 29 Å (Table 1). In these conditions, no improvement of the quality of the fit was observed introducing a second diffusing species in the fitting function (Table 1). The measurements at pH 4.0 and pH 10.0 yielded diffusion coefficient values of 44±3 µm2/s and 31±6 µm2/s, respectively (Table 1) that can be explained in terms of ellipsoidal shaped protein molecules. In these cases, a small amount (2–3%) of slower diffusing molecules (D2 = 15 µm2/s) resulted also to be present (Table 1), suggesting that the protein might form aggregates.


Correlation spectroscopy and molecular dynamics simulations to study the structural features of proteins.

Varriale A, Marabotti A, Mei G, Staiano M, D'Auria S - PLoS ONE (2013)

Fluorescence correlation spectroscopy measurements.Autocorrelation spectra and residual distribution (inset) of MalE2 at pH 7.0 (panel A), pH 4.0 (panel B) and pH 10.0 (panel C). The best fitting of the data was obtained by the Equation 1.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3672191&req=5

pone-0064840-g001: Fluorescence correlation spectroscopy measurements.Autocorrelation spectra and residual distribution (inset) of MalE2 at pH 7.0 (panel A), pH 4.0 (panel B) and pH 10.0 (panel C). The best fitting of the data was obtained by the Equation 1.
Mentions: The correlation function observed in the FCS measurements on MalE2-AL488 in its native state at neutral pH (panel 1A) and at pH 4.0 (panel 1B ) and 10.0 (panel 1C) is shown in Figure 1. The data were analyzed by different models and the best fitting function resulted to be Equation 1, which takes into account the possibility to have one or two species in solution. In particular, the MalE2-AL488 diffusion coefficient at pH 7 was found to be D1 = 84±4 µm2/s, a value compatible with a spherical-shaped molecule with a radius of about 29 Å (Table 1). In these conditions, no improvement of the quality of the fit was observed introducing a second diffusing species in the fitting function (Table 1). The measurements at pH 4.0 and pH 10.0 yielded diffusion coefficient values of 44±3 µm2/s and 31±6 µm2/s, respectively (Table 1) that can be explained in terms of ellipsoidal shaped protein molecules. In these cases, a small amount (2–3%) of slower diffusing molecules (D2 = 15 µm2/s) resulted also to be present (Table 1), suggesting that the protein might form aggregates.

Bottom Line: Our results showed that keeping temperature constant, the protein diffusion coefficient decreased from 84±4 µm(2)/s to 44±3 µm(2)/s when pH was changed from 7.0 to 4.0.An even more marked decrease of the MalE2 diffusion coefficient (31±3 µm(2)/s) was registered when pH was raised from 7.0 to 10.0.The obtained fluorescence correlation data, corroborated by circular dichroism, fluorescence emission and light-scattering experiments, are discussed together with the MD simulations results.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Molecular Sensing, IBP-CNR, Naples, Italy. a.varriale@ibp.cnr.it

ABSTRACT
In this work, we used a combination of fluorescence correlation spectroscopy (FCS) and molecular dynamics (MD) simulation methodologies to acquire structural information on pH-induced unfolding of the maltotriose-binding protein from Thermus thermophilus (MalE2). FCS has emerged as a powerful technique for characterizing the dynamics of molecules and it is, in fact, used to study molecular diffusion on timescale of microsecond and longer. Our results showed that keeping temperature constant, the protein diffusion coefficient decreased from 84±4 µm(2)/s to 44±3 µm(2)/s when pH was changed from 7.0 to 4.0. An even more marked decrease of the MalE2 diffusion coefficient (31±3 µm(2)/s) was registered when pH was raised from 7.0 to 10.0. According to the size of MalE2 (a monomeric protein with a molecular weight of 43 kDa) as well as of its globular native shape, the values of 44 µm(2)/s and 31 µm(2)/s could be ascribed to deformations of the protein structure, which enhances its propensity to form aggregates at extreme pH values. The obtained fluorescence correlation data, corroborated by circular dichroism, fluorescence emission and light-scattering experiments, are discussed together with the MD simulations results.

Show MeSH